Gene correction therapy is one of the most important application directions in regenerative medicine. Scientists are exploring various approaches to introduce targeted mutations/corrections to pluripotent stem cells (PSCs), to establish disease models and/or to develop therapeutic agents. Emerging technologies such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeats) associated protein 9 (Cas9) have enabled high efficient gene knockout (KO) in pluripotent stem cells in humans and model animal species. Indeed these meganucleases especially Cas9 are becoming the mainstream choice for gene targeting in stem cells and in transgenic animal production. However, the knock-in efficiency remains to be further improved, even in the presence of these meganucleases. We hypothesize that inhibiting non-homologous end joining (NHEJ) and/or enhancing the homology directed repair (HDR) via small molecules will improve the meganuclease mediated knock-in efficiency in pluripotent stem cells. In this project, we will test the feasibility by screening smll molecules using a reporter cell line to identify candidate compounds, followed by validation of the efficacy of these compounds in human iPSCs. In Aim 1, we will first establish a reporter cell line that will express luciferase upon successful Ca9 mediated homologous recombination (HR). We will then screen ~13,700 small molecule compounds on this reporter cell line. After identifying candidate compounds in Aim1, we will validate their efficacy on human induced PSCs (iPSCs) in Aim 2. We will work to determine the Cas9 mediated knock-in efficiency in gene correction on multiple human iPSC lines using candidate compounds identified in Aim 1. This proposal aims to address a bottleneck problem in regenerative medicine (i.e. low knock-in efficiency in PSCs). Its success will have significant impacts on the entire field, as a majority o stem cell based therapy will require targeted gene modifications. Notably, it may even enable multiplex gene corrections in human PSCs.